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IFITM3 Regulates NCAPG Through Phosphorylation to in Uence The IFITM3 Regulates NCAPG Through Phosphorylation to Inuence the Invasion and Metastasis of HCC Weiwei Liu ( [email protected] ) Nanchang University https://orcid.org/0000-0002-5850-8769 Rongguiyi Zhang Nanchang University Enliang Li Nanchang University Second Aliated Hospital Linquan Wu Nanchang University Second Aliated Hospital Jiakun Wang Nanchang University Second Aliated Hospital Research article Keywords: IFITM3, NCAPG, HCC, invasion and metastasis, phosphorylation modication Posted Date: September 22nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-54017/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Abstract Background: Several studies have demonstrated that the expression of IFITM3 and NCAPG is closely related to the prognosis of various tumors. However, the mechanism of action of these two is not yet clear. In this study, we have explored the mechanism of action of IFITM3 and NCAPG in the promotion of the invasion and metastasis of hepatocellular carcinoma (HCC). Methods: Specimens of liver cancer and adjacent tissues from 55 HCC patients at the Department of Hepatobiliary Surgery, Second Aliated Hospital of Nanchang University were collected, and the expression of NCAPG and IFITM3 was determined by qRT-PCR and Western blot. Through the analysis of multiple databases, the relationship between IFITM3 and NCAPG was identied and veried by the CO-IP method. Using SiRNA and plasmids to downregulate and up-regulate IFITM3, we observed the expression of STAT3/CDK1, as well as NCAPG mRNA and protein. After downregulating and up-regulating the expression of IFITM3 and NCAPG, the ability of HCC cells to invade and metastasize was determined using a scratch test and Transwell. After using pathway inhibitors and activators, the expression of NCAPG was observed. Results: According to the database, both IFITM3 and NCAPG were highly expressed in Liver hepatocellular carcinoma. We also conrmed that IFITM3 and NCAPG were up-regulated in HCC tissues and cells. Furthermore, the bioinformatics analysis and CO-IP indicated that there was protein interaction between IFITM3 and NCAPG, and that IFITM3 could regulate NCAPG by phosphorylating it. We further conrmed our observations by retrospective experiments. Reuse of pathway inhibitors and activators indicated that IFITM3 could regulate NCAPG through STAT3/CDK1 to promote the invasion and metastasis of HCC. Finally, animal experiments conrmed that our results were also reproducible in vivo. Conclusion: IFITM3 can regulate NCAPG through STAT3/CDK1 to promote the invasion and metastasis of HCC. 1. Background Liver cancer is a common malignant tumor, and hepatocellular carcinoma (HCC) is the most common type of liver cancer[1, 2]. HCC has a poor prognosis, as it rapidly metastasizes, and by the time diagnosis is made, most of it is already in the advanced stage of cancer[3, 4]. Therefore, the identication of biomarkers of liver cancer and understanding the mechanism of invasion and metastasis of liver cancer needs to be pursued urgently. Interferon-induced transmembrane protein 3 (IFITM3) is a member of the interferon-stimulating gene (ISG) family[5, 6]. Previous studies have reported that IFITM3 is closely related to infections by several viruses[7-9]. For example, studies have shown that restriction of viral entry by IFITM3 inhibits the infectivity of iris virus and Noda virus[10]. Sun et al. have also shown that IFITM3 prevents acute inuenza in mice[11]. The latest research has also reported that IFITM3 is closely related to the development of COVID-19[12, 13]. The BAT SARS WIV1 coronavirus uses the ACE2 of a variety of animals Page 2/21 as the receptor and evades IFITM3 through the activation of the membrane fusion protein TMPRSS2[14]. In addition to being closely related to viral infections, the expression of IFITM3 is also signicantly positively correlated with the prognosis of several tumors[13, 15-17]. For example, IFITM3 up-regulates the expression of c-myc through the ERK1/2 signaling pathway to promote the proliferation of liver cancer cells[8]. Although IFITM3 has been proven to be a key gene affecting disease progression in several elds, its inuence in liver cancer has been relatively scarcely studied, and its specic mechanism remains to be understood. The non-SMC condensin I complex subunit G (NCAPG), which organizes the coiled topology of a single chromatid, represents an overexpressed antigen in various types of cancer[18-20]. It helps reorganize chromatin into rod-shaped mitotic chromosomes and ensures the separation of sister chromatids during cell division[21, 22]. In the earliest research, NCAPG was identied as a breeding gene in dairy cows. Subsequent research reported that NCAPG was associated with the progression of various diseases[23, 24]. For example, NCAPG was identied as a risk factor for psoriasis. It was also involved as a key gene in the progression of various tumors[25-27]. For example, NCAPG is overexpressed in colorectal cancer and prostate cancer tissues, and has a close relationship with its prognosis. The latest research has reported that NCAPG plays an extremely important role in HCC, but the specic mechanism of action is still unknown. Protein phosphorylation is an important step in the post-translational modication of proteins and plays an important role in determining the activity of enzymes and other important functional molecules, the delivery of secondary messengers, and the cascade of enzymes[28, 29]. STAT3 is a member of the STAT family. It is an important factor that is involved in the modication of phosphorylation and is an important nuclear transcription factor[30, 31]. STAT3 can also be activated by HBV, HCV, and various oncogene proteins[32]. An unregulated STAT/SOCS signal can also lead to the activation of STAT3, which then regulates the transcription of downstream genes[30, 33, 34]. Hepatocellular carcinoma (HCC) tissues show a signicant overexpression of STAT3, which can result in malignant transformation of hepatocytes and cause cancer. Furthermore, activation of the STAT3 signal and c-Myc, EGFR, TGF, survivin, and VEGF disorders are closely related to the occurrence and development of HCC[35, 32]. Cyclin-dependent kinases (CDK) represent a Ser/Thr kinase system that corresponds to the cell cycle process. It is not only regulated by phosphorylation and dephosphorylation but also is affected by oncogenes and tumor suppressor genes. Therefore, identifying and clarifying the relationship between STAT3/CDK1 and HCC is crucial for the diagnosis and treatment of liver cancer. In our research, we observed that both IFITM3 and NCAPG played an irreplaceable role in the invasion and metastasis of HCC, and IFITM3 could positively regulate NCAPG. Importantly, we discovered for the rst time that IFITM3 could inuence the invasion and metastasis of HCC by changing the level of NCAPG phosphorylation modication. 2. Methods 2.1. Tissue specimen Page 3/21 The study was carried out on 55 patients with HCC diagnosed between 2015 and 2019. Only patients that had undergone hepatectomy without any treatment before surgery, including radiotherapy or chemotherapy, were included in the study. The liver cancer and adjacent tissue specimens were placed in liquid nitrogen immediately upon collection. The study was approved by the Ethics Review Committee of the Second Aliated Hospital of Nanchang University. The procedure followed the ethical standards of the Human Experiment Responsibility Committee (institution and country) and the 1975 “Helsinki Declaration” (revised in 2008). Informed consent was obtained from all the patients before enrollment. 2.2. Cell culture The hepatocyte cell line (HL-7702) and four HCC cell lines (SMMC7721, MHCC97H, HCCLM3, and Huh-7) used in this study were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). All cell lines were cultured in Dulbecco’s Modied Eagle Medium (DMEM) (Solarbio, Beijing, China) supplemented with 10% FBS (Biological Industries, Beit-Haemek, Israel), 100 µg/mL streptomycin, and 100 U/mL penicillin, incubated in an incubator containing 5% carbon dioxide. All the experiments used cells in the logarithmic phase of growth. 2.3. Cell transfection The SiRNA and plasmids were obtained from Ruibo (Guangzhou), and the HCC cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientic, Inc), according to the manufacturer’s instructions. All the transfected cells were incubated in complete medium for at least 24 h prior to transfection and rinsed with phosphate buffered saline (PBS, pH 7.4) before transient transfection. 2.4. RNA extraction and qRT-PCR Total RNA was isolated from the cells using Trizol reagent (Invitrogen), according to the manufacturer’s instructions. Reverse transcription (RT) and qRT-PCR were performed using PrimeScript RT kit (Dalian, China, Treasure) and SYBR Prime Script RT PCR kit (Dalian, China, Treasure). The sequences of IFITM3, NCAPG, STAT3, CDK1, and GAPDH are provided in the supplementary information. The result was calculated using the 2-ΔΔCt method. 2.5. Western blot (WB) All the proteins were separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis (SDS- PAGE), transferred to a nitrocellulose membrane (Amersham, USA), and sealed with 5% skim milk at room temperature. The membrane was then treated with reagents containing the rabbit polyclonal antibody IFITM3 (ab109429, 1:1000, Abcam, Cambridge, UK), NCAPG rabbit polyclonal antibody (ab226805, 1:2000, Abcam, Cambridge, UK), the STAT3 polyclonal rabbit antibody (ab68153, 1:1000, Abcam, Page 4/21 Cambridge, UK), CDK1 rabbit polyclonal antibody (ab18, 1:10000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1:2500, Abcam, Cambridge, UK). The treated membrane was then incubated at 4°C overnight, after which it was washed thrice with PBST buffer (PBS buffer containing 0.1% Tween-20) for 10 min. Horse peroxidase-labeled anti-rabbit IgG secondary antibody (ab6721, 1:2000, Abcam, Cambridge, UK) was then added to the membrane and incubated for 1 h at room temperature. The membrane was washed thrice with PBST buffer for 10 min. A photometer (GE, USA) was used to detect immune activity.
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